phd thesis presentation uam for linkedin
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Combined Gene Therapy for GliomasThe linamarase/linamarin/glucose oxidase system as a potential
treatment for glioblastoma in the nude rat brain
Willie Girald-Rosa, PhDAutonomous University of Madrid, SpainMolecular Biology Center Severo Ochoa
Brain Tumors Primary brain tumors
◦ Glioblastoma multiform (GBM) Life expectancy: on average 14 months
Secondary brain tumors Classification of neoplasms affecting
the central nervous system:
WHO
Grade I Grade II Grade III Grade IV(GBM)
Low-grade High-grade
Not anaplasticwell-differentiated Anaplastic
Undifferentiated
Cancer stem cells cause tumour regrowth after apparently successful therapy
Initial GBM Minimal residual Recurrent GBM
Heterogeneous tumorResidual brain
tumor stem cells
Heterogeneous tumor regrowth from remaining tumor stem cells
Surgery, radio & chemo
Time
CSCs are proposed to persist in tumors as a distinct population and cause relapse by given rise
to new tumors
Glioma Cancer Stem cells (GCSCs) Possess characteristics associated with normal neural stem cells GCSCs are tumorigenic Specific Cellular Surface Markers
◦ Glioma Cancer Stem Cells (GCSCs): CD133+ & Nestin
Cancer CellsGCSCs
Glioma Cancer Stem Cell
Tumor recurrence
Conventional therapy targeting
tumor bulk
Tumor RegressionExisting GCSC replicate and differenciate
GCSCs targeted therapy
Eradication of GCSCs
Non-Conventional therapy targeting tumor bulk
Tumor eradication
Casava Plant, Cyanogenesis & lis-gene
Cyanohydrin group
Cyanogenic compound
LinamarinPlant Defense
Manihot esculenta
Epo-linamarase gene IRES pac
pILE 6.9Kb
amp r pBS Ori
pCM
V
U87MG-lis Cell line & CSCs(-5 & -7) lines
GBM tumor
Dissociation
Culturing with mitogens
Flow cytometry CD133+ CellsProliferation
Dissociation
Culturing with mitogens
Establishment of cell lines GCSCs-5 & GCSCs-7
Proliferation
-Removal of Growth factors-Addition of RADifferentiation
Neurophere
U87MGCells
pILE
puromacin
U87MG-lis
Actin
epo-lis-gene
Linamarase expression
Neural stem cells expression markers in neuropheres
To-Pro 3 Nestin
Mix
CD133
Multipotenciality analysis of the CSCs-5
β-III-Tubuline GFAP MixContrast
NG-2 MAP2/GFAP
Tumor generated by orthotopic injection of CSCs-5 cells in immunodeficent rat brain
Stereotaxic surgery
Rat’s brain with glioma
MRI
The linamarase/linamarin/glucose oxidase system
Lis/linReaction
GO/glucose Reaction
Aspergillus niger
The effect of the lis/lin/GO system
Cellular Death is accompained by mitochondrial fission and ATP depletion (Gargini R et.al. 2008).
Cellular Viability Assay to study the effect of lis/lin/GO system in the U87MG-lis in vitro
500 µg/ml linamarin5.5 mEU/ml GO48h @ 37ºC
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; a yellow tetrazole), is reduced to purple formazan in living cells.
Adenovirus type-5 and the E1-deficient replication-deficient adenoviral vector with
the epo-lis gene
ITR ITR0 4020 8060 100mu
E1 E2
E3
E4
Early Genes “E”
L1
L4L2
L5L3
Late Transcription
ITR CMVPromoter
epo Linamarase geneAn
Crucell Company, Holland
Linamarase expression in CSCs from patients
Immunofluorescent detection
Cellular Viability Assay to study the effect of lis/lin/GO system in the
GCSCs in vitro
Induction of Autophagy is associated to lis/lin/GO
•3-methyl Adenine: autophagy inhibitor•ShATG5: RNAi against gene ATG5
• Required for autophagy. Conjugates to ATG12 and associates with isolation membrane to form cup-shaped isolation membrane and autophagosome
Experimental Design
Stereotaxic surgery
Tumor confirmed by MRI
Combined Gene Therapy lis/lin/GO
U87MG-lis implantation
Local & Systemic Treatments
Delivery Systems
Osmotic Pump(OP)Model 2ML1Total Volume 2 ml10 µl/h/7days
Osmotic Pump(OP)Model 2001Total Volume 200 μl10 µl/h/7days
Intratumoral injectionHamilton 10ul 1µl/min
OP
Flow moderator
Catheter
Brain infusion cannula
Strategies to validate the lis/lin/GO system in the nude rat brain
Summary of Results Group II
A: before treatmentB: after treatment Kaplan Meier (p=0,291)
Death after a mean of 14 ± 1.5 days
Local TreatmentLis-Lin-GOx
Group IIIntracranial injections
250 mg/ml lin + 150 EU/ml GO
Death by toxicity
Death by Tumor
Survived
Summary of Results Group V-CRegression of the tumor after treatment
Beforetreatment
14 days post-treatment
MRI T123 days
post-treatment
MRI T223 days
post-treatment
MRI T145 days
post-treatment
Cocktail in OP:400 mg/ml lin20 EU/ml GODelivery rate:~2.25 µl/h
Linamarase & human MHCI detections in vivo in rat’s brain samples after treatment
Short-term toxic effect
M: markerR: rat treated local & systemically for 2 days 0-96 hours: rats treated local & systemically for different time pointsC1R: human lymphoid cells as + control
Conclusions
The killing effect of the lis/lin/GO system against CSCs and U87MG cell are very effective in vitro.
The lis/lin/GO system eliminates small glioma tumors in the brain of nude rats but in the majority of cases is toxic to the animal. The putative therapeutic window must be very narrow.
Systemic treatments, in our conditions, are not therapeutic probably due to the blood-brain barrier.